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CircuLex
Human ISG15 ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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Detailed Protocol
The CycLex Research Product CircuLex Human ISG15 ELISA Kit is provided with removable
strips of wells so the assay can be carried out on separate occasions using only the number of strips
required for the particular determination. Since experimental conditions may vary, an aliquot of the
human ISG15 Standard within the kit, should be included in each assay as a calibrator. Disposable
pipette tips and reagent troughs should be used for all liquid transfers to avoid cross-contamination of
reagents or samples.
Preparation of Working Solutions
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All reagents need to be brought to room temperature prior to the assay. Assay reagents are supplied
ready-to-use, with the exception of 10X Wash Buffer and Human ISG15 Standard.
1. Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of
deionized (distilled) water (ddH2O). Mix well. Store at 4°C for two weeks or -20°C for long-term
storage.
2. Reconstitute Human ISG15 Standard with 1.0 mL of ddH2O. The concentration of the human
ISG15 in vial should be 40 ng/mL, which is referred as a Master Standard of human ISG15.
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Prepare Standard Solutions as follows:
Use the Master Standard to produce a dilution series (below). Mix each tube thoroughly before
the next transfer. The 12 ng/mL standard (Std.1) serves as the highest standard. The Dilution
Buffer serves as the zero standard (Blank).
Volume of Standard
180 µL of Master Standard
300 µL of Std. 1 (12.0 ng/mL)
300 µL of Std. 2 (6.0 ng/mL)
300 µL of Std. 3 (3.0 ng/mL)
300 µL of Std. 4 (1.5 ng/mL)
300 µL of Std. 5 (0.75 ng/mL)
300 µL of Std. 6 (0.375 ng/mL)
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Std.1
Std.2
Std.3
Std.4
Std.5
Std.6
Std.7
Blank
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Dilution Buffer
420 µL
300 µL
300 µL
300 µL
300 µL
300 µL
300 µL
300 µL
Concentration
12.0 ng/mL
6.0 ng/mL
3.0ng/mL
1.5 ng/mL
0.75 ng/mL
0.375 ng/mL
0.188 ng/mL
0 ng/mL
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Note: Do not use a Repeating pipette. Change tips for every dilution. Wet tip with Dilution Buffer
before dispensing. Unused portions of Master Standard should be aliquoted and stored at below
-70°C immediately. Avoid multiple freeze and thaw cycles.
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Sample Preparation
• Optimal dilution of cell conditioned medium for measurement of ISG15 depends on cell number and
IFN-does; require neat to appropriate dilution.
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• Serum and plasma samples require a 10-40 fold dilution depending on ISG15 concentration in sample.
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Cat#: CY-8085
7
Version#: 120710